Oligonucleotides (ONs) that target essential RNA sequences have found numerous recent applications in the modulation of gene expression in cells and as potential therapeutics1, 2. A mechanistic advantage of steric blocking ONs over RNase H-inducing antisense ONs and RISC-inducing siRNA reagents is greater specificity, since binding of an ON to an incorrect RNA is less likely to trigger an undesired off-target biological effect. Secondly, a much wider range of synthetic ON analogues may be used, since there is no requirement for molecular recognition by a host RNA-cleaving enzyme.
Foremost amongst ON analogues useful as steric blocking agents are those with uncharged backbones, such as peptide nucleic acids (PNA)3 and phosphorodiamidate morpholino oligonucleotides (PMO)4. Both PNA and PMO ONs have been used in vivo for RNA targeting applications towards the development of therapeutics5. In cell culture, both PNA and PMO are observed to enter cells only rather poorly and therefore much effort has been expended to develop methods of enhancing cell delivery. Particularly useful has been the attachment of cell penetrating peptides (CPP), such as Penetratin, Tat (48-60), Transportan, and (R-Ahx-R)4 (Ahx=aminohexanoic acid) in the hope that their observed cell translocating power as peptides can be utilized when conjugated to PNA or PMO6-9.
A valuable assay for assessing the activity of steric blocking ONs is that established by Kole and colleagues which involves splice correction of an aberrant thalassemia β-globin intron by a 18-mer synthetic ON (705 site) in the nucleus of HeLa pLuc705 cells and subsequent up-regulation of reporter firefly luciferase10. This assay has a very high dynamic range, such that even very low activity levels can be measured as a positive luminescence read-out. CPP-PNA conjugates targeted to the 705 splice site have been tested in this assay and moderate activity levels have been reported for several different CPPs when the CPP-PNA conjugate is incubated with HeLa pLuc705 cells in the absence of an added transfection agent, whereas PNA alone is inactive11-13. In our laboratories, we found that whereas Tat-PNA or (Lys)8-PNA conjugates required co-incubation with 100 μM chloroquine, an endosomolytic agent, in order to see significant activity in the assay14, 15, activity in the μM range for the (R-Ahx-R)4-PNA and (R-Ahx-R)4-PMO constructs could be obtained in the absence of chloroquine7, 16.
We have also reported a CPP in which six Arg residues were added to the N-terminus of the known CPP Penetratin17, 18. R6-Penetratin (R6Pen) disulfide-conjugated to a PNA complementary to the trans-activation responsive element RNA of HIV-1 showed significant activity in a HeLa cell luciferase reporter assay of inhibition of Tat-dependent trans-activation that required nuclear delivery and binding to TAR RNA in order to inhibit luciferase expression18.
Duchenne muscular dystrophy (DMD) is an X-linked muscle disorder caused mainly by nonsense or frame-shift mutations in the dystrophin gene, occurring with a frequency of about 1 in 3500 live male births and potential therapies are badly needed29. DMD patients suffer from severe, progressive muscle wasting, whereas the milder Becker muscular dystrophy is caused by in-frame deletions resulting in expression of a shortened but partially functional protein. Sequence-specific antisense oligonucleotides (ON) have been shown to induce targeted exon skipping to correct the reading frame of mutated dystrophin mRNA such that shorter dystrophin forms are produced with activity similar to that of Becker muscular dystrophy30,31. Studies have been carried out in cell models, in an mdx dystrophic mouse model containing a nonsense mutation in exon 2331-33, and in a dog model that have shown outstanding promise for the exon skipping approach. Biological activity is achieved as a result of binding of the ON to the dystrophin pre-mRNA in the muscle cell nuclei to cause alteration of splicing patterns by a “steric block” mechanism.
Patients with DMD often suffer from degeneration of cardiac muscle, leading to forms of heart disease such as cardiomyopathy and X-linked dilated cardiomyopathy. Thus, CPPs that allow improved expression of functional or partially functional dystrophin in cardiac muscle are needed.
Antisense oligonucleotides are currently the most promising therapeutic intervention for Duchenne muscular dystrophy. Antisense oligonucleotides modulate dystrophin pre-mRNA splicing, thereby specifically restoring the dystrophin reading frame and generating a truncated but semi-functional dystrophin protein. Challenges in the development of this approach are the relatively poor systemic antisense oligonucleotide delivery and inefficient dystrophin correction in affected non-skeletal muscle tissues, including the heart.
One of the most important factors determining the efficiency of exon skipping is the ON chemistry. The most widely used has been 2′-O-methyl phosphorothioate (2′OMePS). This backbone was initially tested in a Phase I clinical trial targeting exon 51 of dystrophin pre-mRNA in DMD patients involving intramuscular injection34. A similar Phase I trial was carried out using a phosphorodiamidate morpholino oligonucleotide (PMO)35. Phase II clinical trials involving systemic delivery in DMD patients have recently been completed for both 2′OMePS (Goermans N. M. et al (2011) New England J. Med., 364, 1513-1522) and PMO chemistries (Cirak, S. et al (2011) The Lancet, doi:10.1016/S0140-6736(11)60756-3). Studies in mice have suggested higher levels of exon skipping and restoration of dystrophin expression using PMO compared to 2′OMePS35. PMOs are non-ionic molecules and are considered less likely to form unwanted interactions with the intracellular molecules of target cells.
Yin and Wood have examined another non-ionic analogue called peptide nucleic acids (PNA) by intramuscular injection into mdx mice and found significant induction of exon skipping and dystrophin production23. Both PMO and PNA are considered non-toxic ON analogues with high sequence specificity that have significant potential for pharmaceutical development. Exon-skipping PMO has been shown to be well tolerated in mice to 960 mg/kg dosage (Sazani, P. et al (2011) Int, J. Toxocol, 30, 322-333) and to 320 mg/kg in monkeys (Sazani, P. et al (2011) Int. J. Toxicol, 30, 313-321).
Several research groups have been working on the design of CPPs (sometimes called membrane translocating peptides) that when conjugated to non-ionic ONs (such as PNA or PMO) aid their delivery into cells (but not ionic types) and hence boost biological activity of the ON. In the case of PMO, a peptide has been disclosed containing both natural and non-natural amino acids (R-Ahx-R)4-Ahx-B that when conjugated to PMO results in higher levels of steric block activity in a number of cell and in vivo models36. This has been investigated in mouse mdx DMD studies37.
In order to be useful for in vivo applications, it is preferred that CPPs demonstrate effective penetration of the cell and nuclear membranes, particularly when attached to a cargo such as PNA or PMO, in order to enable efficacious splice correction, e.g. EC50 about 0.90 μM or less as measured by the splice correction luciferase assay of Kole et al. Furthermore, the CPP should have good serum stability in order to resist degradation prior to cell penetration. For therapeutic applications CPPs should also have low toxicity.
We previously created a series of CPPs which for conjugation to either a PNA cargo having the 20-mer base sequence GGCCAAACCTCGGCTTACCT [SEQ ID NO:309] (called PNADMD) or a PMO cargo having the 25-mer base sequence GGCCAAACCTCGGCTTACCTGAAAT [SEQ ID NO:310] (called PMODMD). Both PNADMD and PMODMD are commonly used as an oligonucleotide analogue suitable for exon skipping in mdx muscle cells (in vitro) and in mdx mice (in vivo). Pip 5e-PMODMD exhibited exon skipping and restoration of dystrophin expression in differentiated mouse H2K muscle cells and in an mdx mouse model of DMD, including induction of dystrophin production in heart muscle. Pip 5e has the sequence: RXRRBRRXRILFQYRXRBRXRBC [SEQ ID NO:1] and is disclosed in WO2009/147368 and in Yin et al (Molecular Therapy Vol. 19, No. 7 1295-1303, July 2011). The sequence of the Pip 5e peptide can be broken down into three domains, two Arginine rich domains (RXRRBRRXR [SEQ ID NO: 782] and RXRBRXRB) [SEQ ID NO: 772] and a central hydrophobic core (ILFQY) [SEQ ID NO: 799]. Pip-5e was found to have good activity in delivery of PMODMD into heart muscle.
Wu et al (Nucleic Acids Research, 2007, Vol. 35, No. 15 5182-5191) reported that CPP-PMOF (F represents a 3′-carboxyfluorescein tag) conjugates in which X or B residues had been inserted into an oligo-R sequence increased splice correction activity and cell uptake and aided serum binding but that the conjugates did not enter cells as efficiently as R8- and R9-conjugates. They also reported that the number of X residues affects both nuclear antisense activity and toxicity of conjugates, with peptides of greater than 5 X residues exhibiting time and concentration dependent toxicity at 60 μM in cell lines. They suggest keeping the number of X residues to less than 5 to reduce toxicity. They also reported that reducing the number of RX or RB repeats in (RX)n or (RB)n reduces cell uptake and reduces splice correcting ability (in HeLa cell assay).
Abes et al (Nucleic Acids Research, 2008, 36, 6353-6354) discuss CPP molecules having an (RXR)n-PMO structure and reports that of several spacer (X) molecules tested, a linear C4 (Abu), C6 (Ahx), or C8 (Acy) spacer is most effective.
Saleh et al (Bioconjugate Chem. Vol. 21, No. 10 1902-1911 (2010)) reported that increasing the number of Arginine residues in a linear (RXR)n arrangement to 12 and 16 for conjugates with PNA increases the splicing correction ability in a HeLa cell assay, but also increased cell toxicity. Chain branching (2 or 4 branches) did not result in improved activity. 2-chain branches and some 4-chain branches were tolerated for 12 and 16 Arginine constructs, but not for 8 Arginine constructs.
Thus, increasing the number of spacer residues, such as X, appears to increase toxicity and reduce efficiency of cell entry compared to oligoR peptides, but can also increase splice correction activity. On the other hand a high number of R residues appears to lead to increased cell entry efficiency but also increased toxicity, which is undesirable. Thus, CPPs are required that provide a balance of good cell entry efficiency and low toxicity. In addition it is desirable for the CPP to show favourable properties in vivo, such as directing high exon skipping and dystrophin production in the mouse model, mdx, in all muscle types, including heart.